Cosmetic compositions containing lactate dehydrogenase inhibitors

ABSTRACT

Inhibitors of lactate dehydrogenase stimulate keratinocyte proliferation and collagen synthesis in cutaneous tissues. The inhibitors are used preferably with certain co-active ingredients such as pyruvic acid, acetic acid, acetoacetic acid, β-hydroxybutyric acid, a Krebs cycle pathway metabolite, an aliphatic saturated or an unsaturated fatty acid containing from 8 to 26 carbon atoms, an ω-hydroxy acid containing from 22 to 34 carbon atoms, glutamic acid, glutamine, valine, alanine, leucine, and mixtures thereof.

FIELD OF THE INVENTION

The invention relates to compositions for topical application to humanskin, hair, or nails, which compositions contain an inhibitor of lactatedehydrogenase and to methods of using the compositions for treatment andconditioning of skin, hair, or nails.

BACKGROUND OF THE INVENTION

In recent years cosmetic compositions which improve the appearance ofskin have become popular with consumers. There is at the present time ademand for cosmetic compositions which counteract or prevent the visiblesigns of aged and/or dry skin.

Skin cell proliferation is a process required for growth and repair, andit decreases with aging or photodamage. The synthesis of collagen (apredominant skin protein) also decreases with aging or photodamage. Thepresent invention is based at least in part on the discovery thatincreased skin cell proliferation and collagen synthesis, which, inturn, is associated with improved condition and appearance of the skin,can be attained by incorporating a lactate dehydrogenase inhibitor intotopical treatment compositions.

Cutaneous tissues (skin cells, hair follicles, nails) obtain glucose,(the source of bioenergy) predominantly from blood. Glucose is thenenzymatically degraded to pyruvate. Pyruvate is subsequently metabolizedwithin the cutaneous tissues via at least three routes. In the firstroute, pyruvate is oxidized to form the acetyl group of acetyl-coenzymeA, which is then oxidized completely to CO₂ and H₂ O via the Krebscycle. In the second route, pyruvate is reduced to lactate. In the thirdroute, pyruvate serves as a precursor for biosynthesis of three aminoacids (valine, alanine, and leucine).

The second route is inefficient in terms of energy production.Specifically, conversion of one glucose molecule to lactate yields onlyabout 5-7% of the total energy that can be set free if the glucose isoxidized completely via Krebs cycle to CO₂ and H₂ O.

Although it is known that pyruvate is metabolized in cutaneous tissuevia at least three routes and that the conversion of pyruvate to lactateappears to be a predominant route, it is not known exactly what fractionof pyruvate is metabolized via each of the three routes. Therelationship between aging and energy generation in cutaneous tissues isnot entirely understood, although it has been suggested that cellularbioenergy loss accompanies aging. See e.g., Linnane, Anthony W., et al."Mitochondrial DNA Mutation and the Ageing Process: Bioenergy andPharmacological Intervention", Mutation Research, Vol. 275 (1992) pp.195-208.

Accordingly, it is an object of the present invention to providecompositions for treatment of skin, hair, or nails.

It is another object of the present invention to provide a skintreatment composition containing an inhibitor of lactate dehydrogenase.

It is another object of the invention to provide a method for treatingor preventing the appearance of wrinkled, flaky, aged, photodamaged skinor skin disorders.

These and other objects of the invention will become more apparent fromthe detailed description and examples which follow.

SUMMARY OF THE INVENTION

The above objects are attained by the present invention which includes,in part, a novel composition for topical application to human skin, hairor nails, the composition comprising:

(i) from about 0.001% to about 20% of an inhibitor of lactatedehydrogenase; selected from the group consisting of oxamic acid, anN-substituted oxamic acid, β-chlorolactic acid, thiolactic acid,1,6-dihydro NAD, 4-pyridyl pyruvic acid, quinoline-2-carboxylic acid,suramin sodium, isoquinoline, papaverine, berberine, and mixturesthereof; and

(ii) cosmetically acceptable vehicle for the lactate dehydrogenaseinhibitor.

In the preferred embodiment of the invention, in order to maximize theefficacy of the inventive compositions, a co-active ingredient isincluded which is selected from the group consisting of pyruvic acid,acetic acid, acetoacetic acid, β-hydroxybutyric acid, a Krebs cyclepathway metabolite, an aliphatic saturated or an unsaturated fatty acidcontaining from 8 to 28 carbon atoms, an ω-hydroxy acid containing from22 to 34 carbon atoms, glutamic acid, glutamine, valine, alanine,leucine, and mixtures thereof. Most preferably, the co-active ingredientis selected from the group consisting of Krebs cycle metabolites, i.e.,citric acid, isocitric acid, cis-aconitic acid, 2-ketoglutaric acid,succinic acid, fumaric acid, malic acid, oxaloacetic acid, and mixturesthereof. D, DL or L-stereoisomeric forms of citric, isocitric and malicacid may be present. L-stereoform is most preferred in order to maximizethe efficacy of the inventive compositions.

The present invention also includes a method of treating and improvingthe condition of skin, hair and nails which method includes applying tothe skin, hair, or nails a composition containing the lactatedehydrogenase inhibitor described above in a cosmetically acceptablevehicle.

A particularly preferred use of the inventive compositions is forimproving or preventing the appearance of wrinkled, flaky, aged,photodamaged skin, the appearance of age spots, and treating skindisorders. The compositions according to the invention are intended fortopical application to mammalian skin which is already in dry, flaky,wrinkled, aged, photodamaged condition or which suffers from a skindisorder, or, in the alternative, the inventive compositions may beapplied prophylactically to normal healthy skin to prevent or reduce thedeteriorative changes.

DETAILED DESCRIPTION OF THE INVENTION Lactate Dehydrogenase Inhibitor

An inhibitor of lactate dehydrogenase is the essential ingredient of theinventive compositions.

Lactate dehydrogenase appears in animal tissues as five differentisozymes. All the lactate dehydrogenase isozymes contain fourpolypeptide chains, each of molecular weight about 33,500, but the fiveisozymes contain varying ratios of two kinds of polypeptide chains whichdiffer in composition and sequence. The A chains (also designated M formuscle) and the B chains (also designated H for heart) are coded by twodifferent genes. In skeletal muscle the lactate dehydrogenase isozymecontains four A chains. In heart the predominant isozyme contains four Bchains, The lactate dehydrogenase isozymes in other tissues are amixture of five possible forms which may be designated A4, A3B, A2B2,AB3 and B4. The different lactate dehydrogenase isozymes differsignificantly. For instance, the properties of LDH isozyme A4 favorrapid reduction of pyruvate to lactate in skeletal muscle, whereas theproperties of isozyme B4 tend to favor rapid oxidation of lactate topyruvate in the heart. Some evidence exists that the predominant isozymefound in skin is A4, although other isozymes are present at least tosome extent. See Nishitani, Koji et al., "Lactate Dehydrogenase IsozymePatterns of Normal Human Fibroblasts and their In Vitro-transformedCounterparts Obtained by Treatment with Co-60 Gamma-Rays, SV40 or4-Nitroquinoline 1-oxide", Gann, 72, April 1981, pp. 300-304;Fleischmajer, Raul, M. D., et al., "Lactate Dehydrogenase IsozymePatterns in Blister Fluids", The Journal of Investigative Dermatology,Vol, 50, No. 5 (1968), pp. 405-408. It is expected, however, that alactate dehydrogenase inhibitor will inhibit all isozymes of lactatedehydrogenase, at least to some degree.

Suitable lactate dehydrogenase inhibitors are as follows: oxamic acid,an N-substituted oxamic acid, β-chlorolactic acid, thiolactic acid,1,6-dihydro NAD, 4-pyridyl pyruvic acid, quinoline-2-carboxylic acid,suramin sodium, isoquinoline, papaverine, berberine, and mixturesthereof. Examples of N-substituted oxamic acid include N-alkylsubstituted of oxamic acid N-aryl substituted oxamic acid andN,N-alkylaryl substituted oxamic acid (e.g., ethyl N-N-benzyl-methyloxamic acid). Oxamic acid, β-chlorolactic acid, thiolactic acid,quinoline-2-carboxylicacid, suramin sodium, isoquinoline, papaverine,berberine are available from Sigma. Suramin sodium is available fromBayer.

1,6-dihydro NAD may be synthesized as described by Godtfredson et al.,"1,6-dihydro-NAD as an Humidity Induced lactate dehydrogenase inhibitorin NADH Preparations", Carlsberg. Res. Comm., 43 (3), 171-175 (1978),which is incorporated by reference herein. 4-pyridyl pyruvic acid may besynthesized as described by Sheffield et al., in "Synthesis of Some 4pyridyl pyruvic acids as potential inhibitors", J. Chem Soc. PerkinTrans. I, No. 20, 1972, pp. 2506-2512, which is incorporated byreference herein. N-substituted oxamic acid may be synthesized asdescribed by Meany et al. in "N-substituted Oxamates as Inhibitors ofLactate Dehydrogenase", S. Afr. J. Sci, vol. 77, No. 12, pp. 566-568,1981, which is incorporated by reference herein.

The lactate dehydrogenase inhibitor is present in the inventivecompositions in an amount of from about 0.001% to about 20% by weight ofthe composition. Preferably, in order to maximize efficacy and tominimize cost, the amount is from about 1% to about 10%, most preferablyfrom about 4% to about 8%.

The suitability of a particular inhibitor for treatment of a particularcutaneous tissue may be determined by measuring the activity of lactatedehydrogenase in the presence of the inhibitor, as described in Example1 herein.

In the preferred embodiment of the invention, the inventive compositionsare skin treatment compositions, wherein the lactate dehydrogenaseinhibitor is selected from the group consisting of oxamic acid,thiolactic acid, and mixtures thereof, in order to maximize efficacy andstability and to minimize cost. For these reasons, most preferably, thelactate dehydrogenase inhibitor in skin treatment compositions is oxamicacid.

Cosmetically Acceptable Vehicle

The composition according to the invention also comprises a cosmeticallyacceptable vehicle to act as a dilutant, dispersant or carrier for theactive components in the composition, so as to facilitate theirdistribution when the composition is applied to the skin, hair and/ornails.

Vehicles other than water can include liquid or solid emollients,solvents, humectants, thickeners and powders. An especially preferrednonaqueous carrier is a polydimethyl siloxane and/or a polydimethylphenyl siloxane. Silicones of this invention may be those withviscosities ranging anywhere from about 10 to 10,000,000 centistokes at25° C. Especially desirable are mixtures of low and high viscositysilicones. These silicones are available from the General ElectricCompany under trademarks Vicasil, SE and SF and from the Dow CorningCompany under the 200 and 550 Series. Amounts of silicone which can beutilized in the compositions of this invention range anywhere from 5 to95%, preferably from 25 to 90% by weight of the composition.

The cosmetically acceptable vehicle will usually form from 5 to 99.9%,preferably from 25 to 80% by weight of the emulsion, and can, in theabsence of other cosmetic adjuncts, form the balance of the composition.

Preferred Co-active Ingredient

According to the present invention, the performance of the lactatedehydrogenase inhibitor can be substantially enhanced by the inclusioninto the composition of a co-active ingredient selected from the groupconsisting of pyruvic acid, acetic acid, acetoacetic acid,β-hydroxybutyric acid, a Krebs cycle pathway metabolite, an aliphaticsaturated or an unsaturated fatty acid containing from 8 to 26 carbonatoms, an ω-hydroxy acid containing from 22 to 34 carbon atoms, glutamicacid, glutamine, valine, alanine, leucine, and mixtures thereof.

The term "Krebs cycle pathway metabolite" as used herein means citricacid, isocitric acid, cis-aconitic acid, 2-ketoglutaric acid, succinicacid, fumaric acid, malic acid, oxaloacetic acid, and mixtures thereof.

Suitable examples of saturated or unsaturated fatty acid containing from8 to 26 carbon atoms include, but are not limited to lauric, palmiticand linoleic acids. Suitable examples of ω-hydroxy acid containing from22 to 34 carbon atoms include but are not limited to 22-hydroxydocosanoic acid; 23-hydroxy tricosanoic acid; 24-hydroxy tetracosanoicacid; 30-hydroxy triacontanoic acid; 31-hydroxy untriacontanoic acid;32-hydroxy dotriacontanoic acid; 33-hydroxy tritriacontanoic acid.

The preferred co-active ingredient included in the inventivecompositions is selected from pyruvic acid, acetic acid, acetoaceticacid, β-hydroxybutyric acid, a Krebs cycle pathway metabolite, andmixtures thereof, and most preferably is selected from Krebs cyclepathway metabolites, particularly 2-ketoglutaric acid, L-malic acid, andmixtures thereof, in order to optimize efficacy and stabilty at aminimum cost.

The preferred co-active ingredient is present in the inventivecompositions in an amount of from about 0.01% to about 20% by weight ofthe composition.

Preferably, in order to maximize efficacy and to minimize cost, theamount is from about 1% to about 10%, most preferably from about 4% toabout 8%.

pH of the Composition

The pH of the inventive compositions is important in order to attain thepenetration of active ingredients into skin. Generally, the pH of theinventive compositions is in a range of from about 3 to about 8.Preferably, in order to maximize penetration, the pH is in the range offrom about 3 to about 5, most preferably pH is in the range of from 3.5to 4.5.

Several of lactate dehydrogenase inhibitors and the co-activeingredients used herein are acids. It should be noted that in order tofunction as a lactate dehydrogenase inhibitor some of these acids haveto be in a salt form. However, depending on the pH of the compositionand the pKa of a particular acid employed, the salt may be present inthe composition in the form of an acid, or in the form of an acid/saltmixture. It should be understood that the relative fractions of salt andacid in the salt/acid mixture in the composition may vary once thecomposition is applied to skin, depending on the differences between thepH of skin (typically, slightly acidic, around 6-7, but varies fromindividual to individual and depends on skin condition) and the pH ofthe composition. It should also be understood that if a compoundsatisfies a lactate dehydrogenase inhibition test conducted at pH7.0-7.5 as described in Example 1, the compound is suitable forinclusion in the composition. For instance, if the pH of the compositionis about 3-4, a major fraction or all of oxamate salt in the compositionis present in the composition in the form of oxamic acid. However, suchcomposition is still within the scope of the present invention becauseafter application to skin, at least some of the acid will be convertedinto a salt and will be able to function as a lactate dehydrogenaseinhibitor. Put another way, if a compound present in the compositionsatisfies a lactate dehydrogenase inhibition test at pH 7.0-7.5, thecomposition is within the scope of the invention regardless of the pH ofthe composition.

For the sake of illustration, the following active and co-activeingredients may be present in the inventive compositions in the form ofa salt: oxamic acid, N-substituted oxamic acid, β-chlorolactic acid,thiolactic acid, 4-pyridyl pyruvic acid, quinoline-2-carboxylic acid,pyruvic acid, acetic acid, acetoacetic acid, β-hydroxybutyric acid, analiphatic saturated or an unsaturated fatty acid containing from 8 to 26carbon atoms, an ω-hydroxy acid containing from 22 to 34 carbon atoms,glutamic acid, citric acid, isocitric acid, cis-aconitic acid,2-ketoglutaric acid, succinic acid, fumaric acid, malic acid,oxaloacetic acid.

Suitable salts which may be present in the composition include but arenot limited to sodium, potassium, ammonium, triethanolamine, calcium,lithium salts. The salts may be obtained commercially or they may beprepared by methods known in the art, e.g., neutralizing an acid with asuitable base, such as hydroxide bases of ammonium, potassium, sodium.

Optional Skin Benefit Materials and Cosmetic Adjuncts

An oil or oily material may be present, together with an emulsifier toprovide either a water-in-oil emulsion or an oil-in-water emulsion,depending largely on the average hydrophilic-lipophilic balance (HLB) ofthe emulsifier employed.

Various types of active ingredients may be present in cosmeticcompositions of the present invention. Actives are defined as skin orhair benefit agents other than emollients and other than ingredientsthat merely improve the physical characteristics of the composition.Although not limited to this category, general examples includesunscreens, tanning agents, skin anti-wrinkling agents, anti-dandruffagents, hair conditioners and hair growth stimulants.

Sunscreens include those materials commonly employed to blockultraviolet light. Illustrative compounds are the derivatives of PABA,cinnamate and salicylate. For example, octyl methoxycinnamate and2-hydroxy-4-methoxy benzophenone (also known as oxybenzone) can be used.Octyl methoxy-cinnamate and 2-hydroxy-4-methoxy benzophenone arecommercially available under the trademarks, Parsol MCX andBenzophenone-3, respectively. The exact amount of sunscreen employed inthe emulsions can vary depending upon the degree of protection desiredfrom the sun's UV radiation.

A preferred optional active ingredient to be included in the inventivecomposition are ceramides which play an important role in the productionand maintenance of the water permeability barrier of the skin. Suitableceramides and synthetic analogues thereof are disclosed in EuropeanPatent Application No. 534 286, European Patent Application No. 282 816,European Patent Application No. 227 994, U.S. Pat. No. 5,175,321, U.S.Pat. No. 4,985,547, U.S. Pat. No. 5,028,416, U.S. Pat. No. 5,071,971,Japanese Patent Application No. 63192703, U.S. Pat. No. 4,468,519, andU.S. Pat. No. 4,950,688, all U.S. patents are incorporated by referenceherein. Caramides or their synthetic analogues may be present in theinventive compositions at a level of from about 0.00001% to about 5%,preferably from about 0.0001% to about 1%, optimally from about 0.01% to0.5%.

Another preferred optional ingredient is selected from essential fattyacids (EFAs), i.e., those fatty acids which are essential for the plasmamembrane formation of all cells. In keratinocytes EFA deficiency makescells hyperproliferative. Supplementation of EFA corrects this. EFAsalso enhance lipid biosynthesis of epidermis and provide lipids for thebarrier formation of the epidermis. The essential fatty acids arepreferably chosen from linoleic acid, γ-linolenic acid, homo-γ-linolenicaid, columbinic acid, eicosa-(n-6,9,13)-trienoic acid, arachidonic acid,γ-linolenic acid, timnodonic acid, hexaenoic acid and mixtures thereof.A good suitable source of essential fatty acids is sunflower oil orprimrose oil.

The inventive compositions may include hydroxyacids. The hydroxy acidcan be chosen from α-hydroxy acids, β-hydroxyacids, otherhydroxycarboxylic acids (e.g., dihydroxycarboxylic acid,hydroxy-dicarboxylic, hydroxytricarboxylic) and mixtures thereof orcombination of their stereoisomers (DL, D or L).

Preferably, the hydroxy acid (ii) is chosen from α-hydroxy acids havingthe following general structure: ##STR1## where M is H-- or CH₃ (C_(f)H_(g))_(h) --,

f is an integer of from 1 to 27,

g is an integer of from 2 to 54, and

h is 0 or 1.

Even more preferably, the hydroxy acid is chosen from 2-hydroxyoctanoicacid, hydroxylauric lactic acid, and glycolic acid, and mixturesthereof. When stereoisomers exist, L-isomer is most preferred.

The keto acids can be chosen from α-keto acids, β-keto acids andmixtures thereof.

A particularly preferred α-keto acid is 2-keto octanoic acid.

Preferably the amount of the hydroxy acid component (ii) present in thecomposition according to the invention is from 0.01 to 20%, morepreferably from 0.05 to 10%. and most preferably from 0.1 to 3% byweight.

Surfactants, which are also sometimes designated as emulsifiers, may beincorporated into the cosmetic compositions of the present invention.Surfactants can comprise anywhere from about 0.5% to about 30%,preferably from about 1% to about 15% by weight of the totalcomposition. Surfactants may be cationic, nonionic, anionic, oramphoteric in nature and combinations thereof may be employed.

Illustrative of the nonionic surfactants are alkoxylated compounds basedupon fatty alcohols, fatty acids and sorbitan. These materials areavailable, for instance, from the Shell Chemical Company under the"Neodol" designation. Copolymers of polyoxypropylene-polyoxyethylene,available under the Pluronic trademark sold by the BASF Corporation, aresometimes also useful. Alkyl polyglycosides available from the HenkelCorporation similarly can be utilized for the purposes of thisinvention.

Anionic-type surfactants may include fatty acid soaps, sodium laurylsulphate, sodium lauryl ether sulphate, alkyl benzene sulphonate, monoand/or dialkyl phosphates and sodium fatty acyl isethionate.

Amphoteric surfactants include such materials as dialkylamine oxide andvarious types of betaines (such as cocoamido propyl betaine).

Emollients are often incorporated into cosmetic compositions of thepresent invention. Levels of such emollients may range from about 0.5%to about 50%, preferably between about 5% and 30% by weight of the totalcomposition. Emollients may be classified under such general chemicalcategories as esters, fatty acids and alcohols, polyols andhydrocarbons.

Esters may be mono- or di-esters. Acceptable examples of fatty di-estersinclude dibutyl adipate, diethyl sebacate, diisopropyl dimerate, anddioctyl succinate. Acceptable branched chain fatty esters include2-ethyl-hexyl myristate, isopropyl stearate and isostearyl palmitate.Acceptable tribasic acid esters include triisopropyl trilinoleate andtrilauryl citrate. Acceptable straight chain fatty esters include laurylpalmitate, myristyl lactate, oleyl eurcate and stearyl oleate. Preferredesters include coco-caprylate/caprate (a blend of coco-caprylate andcoco-caprate), propylene glycol myristyl ether acetate, diisopropyladipate and cetyl octanoate.

Suitable fatty alcohols and acids include those compounds having from 10to 20 carbon atoms. Especially preferred are such compounds such ascetyl, myristyl, palmitic and stearyl alcohols and acids.

Among the polyols which may serve as emollients are linear and branchedchain alkyl polyhydroxyl compounds. For example, propylene glycol,sorbitol and glycerin are preferred. Also useful may be polymericpolyols such as polypropylene glycol and polyethylene glycol. Butyleneand propylene glycol are also especially preferred as penetrationenhancers.

Exemplary hydrocarbons which may serve as emollients are those havinghydrocarbon chains anywhere from 12 to 30 carbon atoms. Specificexamples include mineral oil, petroleum jelly, squalene andisoparaffins.

Another category of functional ingredients within the cosmeticcompositions of the present invention are thickeners. A thickener willusually be present in amounts anywhere from 0.1% to 20% by weight,preferably from about 0.5% to 10% by weight of the composition.Exemplary thickeners are cross-linked polyacrylate materials availableunder the trademark Carbopol from the B. F. Goodrich Company. Gums maybe employed such as xanthan, carrageenan, gelatin, karaya, pectin andlocust bean gum. Under certain circumstances the thickening function maybe accomplished by a material also serving as a silicone or emollient.For instance, silicone gums in excess of 10 centistokes and esters suchas glycerol stearate have dual functionality.

Many cosmetic compositions, especially those containing water, must beprotected against the growth of potentially harmful microorganisms.Preservatives are, therefore, necessary. Suitable preservatives includealkyl esters of p-hydroxybenzoic acid, hydantoin derivatives, propionatesalts, and a variety of quaternary ammonium compounds.

Particularly preferred preservatives of this invention are methylparaben, propyl paraben, imidazolidinyl urea, sodium dehydroxyacetateand benzyl alcohol. Preservatives will usually be employed in amountsranging from about 0.5% to 2% by weight of the composition.

Powders may be incorporated into the cosmetic composition of theinvention. These powders include chalk, talc, Fullers earth, kaolin,starch, smectite clays, chemically modified magnesium aluminum silicate,organically modified montmorillonite clay, hydrated aluminum silicate,fumed silica, aluminum starch octenyl succinate and mixtures thereof.

Other adjunct minor components may also be incorporated into thecosmetic compositions. These ingredients may include coloring agents,opacifiers and perfumes. Amounts of these materials may range anywherefrom 0.001% up to 20% by weight of the composition.

Use of the Composition

The composition according to the invention is intended primarily as aproduct for topical application to human skin, especially as an agentfor reducing the permeability to water of the skin, particularly whenthe skin is dry or damaged, in order to reduce moisture loss andgenerally to enhance the quality and flexibility of skin. Thecomposition can also be applied to hair and nails.

In use, a small quantity of the composition, for example from 1 to 5 ml,is applied to exposed areas of the skin, from a suitable container orapplicator and, if necessary, it is then spread over and/or rubbed intothe skin using the hand or fingers or a suitable device.

Product Form and Packaging

The topical skin and/or hair treatment composition of the invention canbe formulated as a lotion having a viscosity of from 4,000 to 10,000mPas, a fluid cream having a viscosity of from 10,000 to 20,000 mPas ora cream having a viscosity of from 20,000 to 100,000 mPas or above. Thecomposition can be packaged in a suitable container to suit itsviscosity and intended use by the consumer. For example, a lotion orfluid cream can be packaged in a bottle or a roll-ball applicator or apropellant-driven aerosol device or a container fitted with a pumpsuitable for finger operation. When the composition is a cream, it cansimply be stored in a non-deformable bottle or squeeze container, suchas a tube or a lidded jar.

The invention accordingly also provides a closed container containingcosmetically acceptable composition as herein defined.

The following specific examples further illustrate the invention but theinvention is not limited thereto.

EXAMPLE 1 Procedure for Testing Lactate Dehydrogenase (LDH) Inhibition

All reagents were purchased from Sigma Chemical Co., St. Louis, Mo.,USA. Sigma procedure No. 500 was modified for our purposes. One mL ofpyruvate substrate (Sigma 500L-1, pH=7.5) was added to a vial containing1 mg NADH (Sigma 340-101). In a 96-well plate, the following componentswere added to each test well: 10 μl pyruvate/NADH mixture, and 10 μlcell lysate. For preparation of cell lysates, neonatal humankeratinocytes or fibroblasts (Clonetic Corp., San Diego, Calif.) werecultured in tissue culture plates (Corning, USA). Cells were scraped inPBS (pH=7.4) and this solution was used as a source of L-lactatedehydrogenase. If such cell lysate was not available, a commercialpreparation of L-lactic dehydrogenase (E.C. 1.1.1.27), Sigma L9889, fromporcine heart could be used in place of the cell lysate. For positivecontrol, the inhibitor was replaced with 10 μl of PBS. For blankdetermination, the lysate or enzyme was replaced with 10 mL of PBS.After mixing, the plate was incubated for 30 minutes at 37° C. Then 20μl of color reagent (Sigma 505-2) was added to each well. After 20minutes at room temperature, 150 μl of 0.4N NaOH was added, and theplate was read for absorbance at 490 nm in a Dynatech MR 7000 MicroplateReader.

To calculate % inhibition, the control activity (CA) was firstdetermined by subtracting the absorbance of the positive control fromthe blank. Then the activity in the presence of the inhibitor (IA) wascalculated by subtracting the appropriate absorbance from the blank. %Inhibition was then calculated as: (CA-IA)/CA!*100%.

                  TABLE 1                                                         ______________________________________                                        Inhibitors of Keratinocyte Lactate Dehydrogenase (% Inhibition)                             CONCENTRATION (mM)                                              Code    Inhibitor   5     2.5    1.25 0.625                                   ______________________________________                                        A       Oxamate     97    93     84   71                                      B       Thio-Lactate                                                                              14     8      8    4                                      ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Inhibitors of Fibroblast Lactate Dehydrogenase (% Inhibition)                                CONCENTRATION (mM)                                             Code    Inhibitor    5      2.5    1.25 0.625                                 ______________________________________                                        A       Oxamate      96     92     87   76                                    B       Thio-Lactate 11     11     8    7                                     C       Glycolate    0      0      0    0                                     D       Malonate     0      0      0    0                                     E       L-lactate    0      0      0    0                                     F       D-lactate    0      0      0    0                                     G       Pyrazole     0      0      0    0                                     H       Methyl pyrazole                                                                            0      0      0    0                                     I       Glycinate    0      0      0    0                                     K       L-malate     0      0      0    0                                     ______________________________________                                    

The results in Tables 1 and 2 demonstrate an effective procedure forascertaining whether a compound inhibits lactate dehydrogenase. Theresults also demonstrate that compounds A and B (Tables 1 and 2) wereeffective inhibitors of LDH, while compounds C-K (Table 2) did notinhibit LDH.

EXAMPLE 2 Effect of LDH Inhibitors on Keratinocyte ProliferationProcedure for Measuring Keratinocyte Proliferation

Normal human epidermal keratinocytes (NHEK) derived from neonatalforeskin were used for all experiments. Media were purchased fromClonetics Corp., San Diego, Calif. Cell stocks frozen in passage 2 weregrown in Keratinocyte Growth Medium (KGM) and passaged when 70-80%confluent. Cells were seeded in 96 well plates (Corning) at a density of7500 cells/well in KGM. After 24 hours, cells were rinsed and dosed withtest compounds in 200 μl of Keratinocyte Basal Medium (KBM). Plates wereincubated for three days at 37° C., 5% CO₂. Cell proliferation wasestimated by assaying for DNA content by a fluorometric method describedby Rago et al. (Analytical Biochemistry, 191:31-34, 1990). Medium wasremoved and cells were rinsed with phosphate buffered saline solution.100 μl of sterile distilled water was added to each well and plates werefrozen at -70° C. for one and a half hours. Plates were thawed for onehour at which time 100 μl of a 20 ug/ml solution of Bisbenzimide H33258fluorochrome (Calbiochem Corp., La Jolla, Calif.) was added to eachwell. The fluorochrome was prepared just prior to use in 10 mM Tris, 1mM EDTA, 4M NaCl, pH 7.4. Plates were read in a Millipore Cytofluorfluorescent plate reader to quantify DNA (excitation 360 nm, emission460 nm). Percent increase over control was calculated fromblank-corrected readings as (treated-untreated)/(untreated)*100%. Eachdata point in the Tables below represents a mean of 5 or 6 replicates.The results that were obtained are summarized in Tables 3 and 4.

                  TABLE 3                                                         ______________________________________                                        Effect of Oxamate on Keratinocyte Proliferation (% Increase Over              Control)                                                                      Concentration (mM)                                                            Test   20      10     5.0   2.5 1.25   0.025                                                                              0.313                             ______________________________________                                        A      0       20     28    --  --     --   --                                B      0       33     48    39  28     --   --                                C      0       0       0     0  --     --   --                                D      --      0      23    32  24     20   18                                E      --      0      20    25  40     19   32                                ______________________________________                                         -- Concentration not tested                                              

                  TABLE 4                                                         ______________________________________                                        Effect of Other LDH Inhibitors on Keratinocyte                                Proliferation (% increase over control)                                                   Concentration (mM)                                                Test    Inhibitor 5       2.5 1.25   0.625                                                                              0.313                               ______________________________________                                        F       Thiolactate                                                                             0       5   27     29   9                                   ______________________________________                                    

The results in Tables 3 and 4 indicate that LDH inhibitors stimulatekeratinocyte proliferation. Test C in Table 3 does not appear to be arepresentative dose response curve of the effect of oxamate onkeratinocyte proliferation because it was the only experiment of thefive in which oxamate did not stimulate keratinocyte proliferation atthe concentrations tested. It is possible that the dose response hasshifted in Test C as a result of the cells having different metabolicrequirements as compared to the cells in the other four experiments.Therefore, if lower concentrations had been tested, there most likelywould have been an effect. The dose response appears to be a "bell shapecurve" response. It should be noted that the response obtained at aparticular concentration differs from test to test (see Table 3), as aresult of the cells in different experiments having different metabolicrequirements. Therefore, various actives should be compared within thesame experiments only.

EXAMPLE 3

The procedure of Example 2 was repeated except that various co-activeingredients were tested for their ability to stimulate keratinocyteproliferation.

The results that were obtained are summarized in Tables 5-9.

                                      TABLE 5                                     __________________________________________________________________________    Effect of L-Malate on Keratinocyte Proliferation                              (% Increase Over Control)                                                     Concentration (mM)                                                            Test                                                                             20 10 5.0                                                                              2.5                                                                              2.0                                                                              1.25                                                                             0.625                                                                            0.313                                                                            0.2                                                                              0.15                                                                             0.02                                                                             0.002                                     __________________________________________________________________________    A  ND 0  25 49 ND 53 53 43 ND 19 ND ND                                        B  ND 26 67 60 ND 78 79 78 ND 89 ND ND                                        C  ND ND 0  0  ND 12 33 28 ND 9  ND ND                                        D  ND ND 20 39 ND 25 36 29 ND 33 ND ND                                        E  ND 256                                                                              ND ND 88 ND ND ND 36 ND 27 34                                        F  86 88 ND ND 21 ND ND ND 19 ND ND ND                                        G  11 59 ND ND 28 ND ND ND 29 ND ND ND                                        __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________    Effect of Citrate on Keratinocyte Proliferation                               (% Increase Over Control)                                                     Concentration (mM)                                                            Test                                                                             10 5.0                                                                              2.5                                                                              2.0                                                                              1.25                                                                             1.0                                                                              0.6                                                                              0.3                                                                              0.2                                                                              0.15                                                                             0.1                                                                              0.02                                                                             0.01                                                                             0.002                               __________________________________________________________________________    A  ND ND ND ND ND ND ND ND 33 ND ND 40 ND 30                                  B  ND ND ND ND ND ND ND ND 7  ND ND 17 ND 0                                   C  ND ND ND ND ND 20 ND ND ND ND 40 ND 25 ND                                  D  0  0  0  ND 18 ND 35 39 ND 18 ND ND ND ND                                  __________________________________________________________________________

                                      TABLE 7                                     __________________________________________________________________________    Effect of Acetate on Keratinocyte Proliferation                               (% Increase Over Control)                                                     Concentration (mM)                                                            Test                                                                             20 10 5.0                                                                              2.5                                                                              2.0                                                                             1.25                                                                             0.625                                                                            0.313                                                                            0.2                                                                             0.156                                                                            0.07                                                                             0.02                                                                             0.002                                    __________________________________________________________________________    A  ND 63       46         43      31 22                                       B  0  0        19         12      ND ND                                       C     0  0  11   2  9  32   13 ND                                             D     ND 18 44   42 57 56   47 50                                             E     ND ND ND   ND ND 24   6  0                                              F     0  7  33   35 34 5.0  ND ND                                             G     0  18 12   24 11 38   ND ND                                             __________________________________________________________________________

                                      TABLE 8                                     __________________________________________________________________________    Effect of Isocitrate on Keratinocyte Proliferation                            (% Increase Over Control)                                                     Concentration (mM)                                                            Test                                                                             10 5.0                                                                             2.5                                                                             2.0                                                                             1.25                                                                             0.625                                                                            0.313                                                                            0.2                                                                             0.156                                                                            0.02                                                                             0.002                                                                            0.0002                                        __________________________________________________________________________    A  0      40         62   63 39 ND                                            B  ND     30         29   30 20 0                                             C  0  18                                                                              32  40 32 40   32                                                     __________________________________________________________________________

                                      TABLE 9                                     __________________________________________________________________________    Effect of Oxaloacetate on Keratinocyte Proliferation                          (% Increase Over Control)                                                     Concentration (mM)                                                            Test                                                                             20 10 5.0                                                                             2.5                                                                             2.0                                                                             1.25                                                                             0.6                                                                             0.3                                                                             0.2                                                                             0.15                                                                             0.02                                                                             0.08                                                                             0.002                                        __________________________________________________________________________    A  ND 110    53       22  15     11                                           B  44 70     16       20  30     ND                                           C     0  35                                                                              40  18 20                                                                              18  13    ND                                              D     52 52                                                                              37  37 48                                                                              63  76    56                                              __________________________________________________________________________

The results in Tables 5-9 indicate that various co-active ingredientswithin the scope of the invention stimulated keratinocyte proliferation.

EXAMPLE 4 Assay for Collagen Synthesis

Neonatal human dermal fibroblasts were purchased from Clonetics Corp.,San Diego, Calif. All materials for cell culture were purchased fromLife Technologies, NY. Cells were maintained in DMEM with 10% fetalbovine serum and used in passages 5-10. Confluent 96-well platescontaining fibroblasts were treated with test materials (0.2 to 20 mM)in serum-free medium for 48 hours. Media was collected and animmunoassay utilizing a monoclonal antibody specific for procollagen 1(MAB 1912, Chemicon, Temecula, Calif.) was performed to estimate theamount of secreted pro-collagen in the medium. This was carried out in aBioDot SF apparatus according to the manufacturer's instructions (BioRadLabs, CA) and the blot was developed using a Vectastain Kit (PK6104,Vector Labs, CA) as per the manufacturer's directions. Color intensitywas quantitated using an Ultroscan XL densitometer (Pharmacia LKB). Foldincrease was calculated as (density of treated)/untreated. The resultsthat were obtained are summarized in Table 10.

                  TABLE 10                                                        ______________________________________                                        Effect of LDH Inhibitors or Co-active Ingredients on Collagen                 Synthesis                                                                     Inhibitor    Maximum fold over Control                                        ______________________________________                                        Oxamate      1.4                                                              Acetate      2.0                                                              Acetoacetate 2.2                                                              Ketoglutarate                                                                              2.2                                                              L-Malate     2.6                                                              Succinate    2.8                                                              Oxaloacetate 3.0                                                              Glutamate    3.6                                                              ______________________________________                                    

EXAMPLE 5

The procedure of Example 2 was employed to test the effect ofcombination of an LDH inhibitor and a co-active ingredient onkeratinocyte proliferation. The results that were obtained aresummarized in Tables 11 and 12.

                  TABLE 11                                                        ______________________________________                                        % Increase Over Control                                                       Oxamate                                                                              Pyruvate (mM)                                                          (mM)   0       0.156  0.313  0.625                                                                              1.25   2.5 5.0                              ______________________________________                                        0              2      0      3    0      7   0                                2.5    0       --     --     --   --     --  --                               5.0    0       10     18     4    15     0   0                                10     0       8      8      11   13     0   0                                15     0       0      0      0    0      0   0                                20     0       0      0      0    0      0   0                                ______________________________________                                         -- Concentration not tested                                              

The results in Table 11 indicate that even when the individualingredients did not result in increase in keratinocyte proliferation, atthe concentration tested in a particular experiment, the combination ofthose ingredients resulted in a significant increase over control.

                  TABLE 12                                                        ______________________________________                                        Malate                                                                        % Increase Over Control                                                       Oxamate                                                                              CONCENTRATION (mM)                                                     (mM)   0       0.156  0.313  0.625                                                                              1.25   2.5 5.0                              ______________________________________                                        0              33     29     36   25     39  20                               0.313  32      --     --     --   --     --  --                               0.625  19      --     --     --   --     --  --                               1.25   40      44     29     44   49     37  20                               2.5    25      65     36     74   61     54  20                               5.0    20      38     38     58   57     51   7                               10      0      --     --     --   --     --  --                               ______________________________________                                         -- Not tested                                                            

The results in Tables 11 and 12 demonstrate that the combination of anLDH inhibitor with a preferred co-active ingredient results insubstantial enhancement of keratinocyte proliferation.

EXAMPLE 6

This example illustrates a high internal phase water-in-oil emulsion inaccordance with the invention.

    ______________________________________                                                            % w/w                                                     ______________________________________                                        Thiolactate           1                                                       Acetate               8                                                       Fully hydrogenated coconut oil                                                                      3.9                                                     Neoceramide having the structure (5)                                                                0.1                                                     Brij 92*              5                                                       Bentone 38            0.5                                                     Preservative          0.3                                                     MgSO.sub.4 7H.sub.2 O 0.3                                                     Butylated hydroxy toluene                                                                           0.01                                                    Perfume               qs                                                      Water                 to 100                                                  ______________________________________                                         *Brij 92 is polyoxyethylene (2) oleyl ether                              

EXAMPLE 7

This example also illustrates a high internal phase water-in-oilemulsion in accordance with the invention in which the formulation ofExample 6 is prepared but with the following changes:

(i) liquid paraffin employed instead of the fully hydrogenated coconutoil, and;

(ii) oxamate is used in place of thiolactate.

EXAMPLE 8

This example also illustrates a high internal phase water-in-oilemulsion in accordance with the invention in which the formulation ofExample 6 is prepared but with the following changes:

β-chlorolactate is used in place of thiolactate,

EXAMPLE 9

This example illustrates an oil-in-water cream containing an lactatedehydrogenase according to the invention.

    ______________________________________                                                         % w/w                                                        ______________________________________                                        Oxamate            4                                                          Acetate            4                                                          Mineral oil        4                                                          Thiolactate        2                                                          Brij 56*           4                                                          Alfol 16RD**       4                                                          Triethanolamine    0.75                                                       Butane-1,3-diol    3                                                          Xanthan Gum        0.3                                                        Preservative       0.4                                                        Perfume            qs                                                         Butylated hydroxy toluene                                                                        0.01                                                       Water              to 100                                                     ______________________________________                                         *Brij 56 is cetyl alcohol POE (10)                                            **Alfol 16RD is cetyl alcohol                                            

EXAMPLES 10 AND 11

The following compositions according to the invention represent lotionswhich can be used in the treatment of dry skin:

    ______________________________________                                                          % w/w                                                                         10    11                                                    ______________________________________                                        L-malate            6.0     4.0                                               Thiolactate         2.0     4.0                                               Perfume             0.1     0.1                                               Hydroxyethyl cellulose                                                                            0.4     0.4                                               Absolute ethanol    25      25                                                p-methyl benzoate   0.2     0.2                                               Sterilized Demineralized Wtaer                                                                    to 100  to 100                                            ______________________________________                                    

EXAMPLE 12

This example illustrates an alcoholic lotion containing a lactatehydrogenase inhibitor according to the invention which is suitable forapplication to nails.

    ______________________________________                                                       % w/w                                                          ______________________________________                                        Oxamate          4                                                            Dimethylsulphoxide                                                                             10                                                           Ethanol          40                                                           Antoxidant       0.1                                                          Perfume          qs                                                           Water            to 100                                                       ______________________________________                                    

EXAMPLES 13 AND 14

The following compositions according to the invention represent lotionswhich can be used in the treatment of dry, unmanageable hair.

    ______________________________________                                                          % w/w                                                                         13    14                                                    ______________________________________                                        Pyruvate            6       8                                                 Oxamate             2       1                                                 Perfume             0.1     0.1                                               Hydroxyethyl cellullose                                                                           0.4     0.4                                               Absolute ethanol    25      25                                                p-methyl benzoate   0.2     0.2                                               Sterilized demineralized water                                                                    to 100  to 100                                            ______________________________________                                    

It should be understood that the specific forms of the invention hereinillustrated and described are intended to be representative only.Changes, including but not limited to those suggested in thisspecification, may be made in the illustrated embodiments withoutdeparting from the clear teachings of the disclosure. Accordingly,reference should be made to the following appended claims in determiningthe full scope of the invention.

What is claimed is:
 1. A composition for topical application to humanskin, the composition comprising:(i) from about 0.001% to about 20% ofan inhibitor of lactate dehydrogenase selected from the group consistingof oxamic acid, an N-substituted oxamic acid, 1,6-dihydro NAD, 4-pyridylpyruvic acid, quinoline-2-carboxylic acid, suramin sodium, isoquinoline,papaverine, berberine, and mixtures thereof; and (ii) from about 0.01%to about 20% by weight of the composition of a co-active ingredientselected from the group consisting of pyruvic acid, acetic acid,acetoacetic acid, β-hydroxybutyric acid, citric acid, isocitric acid,cis-aconitic acid, 2-ketoglutaric acid, succinic acid, fumaric acid,malic acid, oxaloacetic acid, an aliphatic saturated or an unsaturatedfatty acid containing from 8 to 26 carbon atoms, an ω-hydroxy acidcontaining from 22 to 34 carbon atoms, glutamic acid, glutamine, valine,alanine, leucine, and mixtures thereof, (iii) a cosmetically acceptablevehicle for the lactate dehydrogenase inhibitor, wherein the vehiclecomprises water; wherein the pH of the composition is from about 3 toabout 8; and wherein the lactate dehydrogenase inhibitor acts within thehuman skin.
 2. The composition of claim 1 wherein the lactatedehydrogenase inhibitor is present in an amount of from about 0.1% toabout 10% by weight of the composition.
 3. The composition of claim 1wherein the co-active ingredient is selected from the group consistingof pyruvic acid, acetic acid, acetoacetic acid, β-hydroxybutyric acid,citric acid, isocitric acid, cis-aconitic acid, 2-ketoglutaric acid,succinic acid, furmaric acid, malic acid, oxaloacetic acid, and mixturesthereof.
 4. The composition of claim 3 wherein the co-active ingredientis selected from the group consisting of pyruvic acid, acetic acid,2-ketoglutaric acid, L-malic acid, succinic acid, and mixtures thereof.5. The composition of claim 1 wherein the lactate dehydrogenaseinhibitor is selected from the group consisting of oxamic acid,thiolactic acid, and mixtures thereof.
 6. The composition of claim 1wherein the pH of the composition is from about 3 to about
 5. 7. Amethod of treating human skin which comprises applying topically theretothe composition comprising:(i) from about 0.001% to about 20% of aninhibitor of lactate dehydrogenase selected from the group consisting ofoxamic acid, an N-substituted oxamic acid, β-chlorolactic acid,thiolactic acid, 1,6-dihydro NAD, 4-pyridyl pyruvic acid,quinoline-2-carboxylic acid, suramin sodium, isoquinoline, papaverine,berberine, and mixtures thereof; and (ii) a cosmetically acceptablevehicle for the lactate dehydrogenase inhibitor.
 8. A method ofimproving the appearance of wrinkled, flaky, aged, photodamaged skin,the appearance of age spots which comprises applying topically theretothe composition comprising:(i) from about 0.001% to about 20% of aninhibitor of lactate dehydrogenase selected from the group consisting ofoxamic acid, an N-substituted oxamic acid, 1,6-dihydro NAD, 4-pyridylpyruvic acid, quinoline-2-carboxylic acid, suramin sodium, isoquinoline,papaverine, berberine, and mixtures thereof; and (ii) a cosmeticallyacceptable vehicle for the lactate dehydrogenase inhibitor.